SCL27 activates <i>MIR171</i> gene expression in a feedback manner.

<p>(A) qPCR analysis of <i>MIR171a</i>, <i>MIR171b</i> and <i>MIR171c</i> expression in Col, <i>scl6 scl22 scl27</i> and <i>LUC-rSCL27-OX</i> plants. Relative expression levels of <i>MIR171</i> genes were normalized to that of <i>ACTIN2</i>, and the relative expression in WT plants was set as 1. Error bars represent the s.d. (n = 3). Two biological replicates were performed with similar results. (B) <i>35S::6xMYC-rSCL27-GR/scl6 scl22 scl27</i> transgenic plants were treated with DEX (10 µM) or untreated (Mock) for 20 days. Bars = 1 cm. (C) Chlorophyll content of the plants shown in (B). ** indicates <i>p</i> value (Student's t-test) <0.01; Error bars indicate the s.d. (n = 4). (D) Relative expression of <i>PORC</i>, <i>MIR171a</i>, <i>MIR171b</i> and <i>MIR171c</i> in the plants shown in (B). Expression levels were normalized to that of <i>ACTIN2</i>. Expression levels in plants without DEX were set as 1 for each gene. Error bars represent the s.d. (n = 3). Two biological replicates were performed with similar results. (E) Relative activity of the <i>MIR171a</i> promoter. <i>pMIR171a::LUC</i> was transformed into <i>N. benthamiana</i> leaves with or without co-transformation of <i>6xMYC-rSCL27</i>. Relative <i>LUC</i> activities were normalized to the <i>35S::REN</i> internal control. Error bars indicate the s.d. (n = 4). Three biological replicates showed similar results. (F) Schematic diagram of <i>MIR171a</i> promoter regions V (−726 bp to −495 bp), VΙ (−260 bp to −71 bp) and VΙΙ (the precursor of <i>MIR171a</i>), which were used for ChIP experiments. (G) Relative enrichment of <i>MIR171a</i> promoter fragments in the immuno-precipitates. Leaves of 3-week-old Col and <i>6xMYC-rSCL27-OX</i> plants were used for ChIP experiments. The enriched DNA fragments were quantified using qPCR. The <i>β-TUBULIN-2</i> promoter was used as a reference. Error bars indicate the s.d. (n = 3). Similar results were obtained from three independent immuno-precipitation experiments.